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Techniques that quantify molecular endpoints sufficiently sensitive to identify and classify potentially toxic compounds have wide potential for high-throughput in vitro screening. Expression of three genes, RAD51C, TP53 and cystatin A (CSTA), in HEPG2 cells was measured by Q-PCR amplification. In parallel, we developed alternative assays for the same 3 gene signature based on an acridinium-ester chemiluminescent reporter molecule. HEPG2 cells were challenged with eighteen different compounds (n=18) chosen to represent compounds that are genotoxic (n=8), non-genotoxic non-carcinogenic (n=2) or have a less well defined mechanism of action with respect to genotoxicity (n=8). At least one of the three genes displayed dysregulated expression in the majority of compounds tested by Q-PCR and ten compounds changed the CSTA expression significantly. Acridinium-ester labelled probes for the three genes were synthesised and tested. Analytical sensitivity was characterised and suggested a limit of detection generally better than 0.1fmol but often 10-50 attomol. A linear amplification step was optimised and this quantitative method detected statistically significant increases in RAD51C and CSTA expression in agreement with the Q-PCR results, demonstrating the potential of this technology. The broad agreement of the amplified chemiluminescent method and Q-PCR in measuring gene expression suggests wider potential application for this chemiluminescent technology., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The molecular signalling pathways mediating sexual dimorphism have principally been investigated in the gonads, and to a lesser extent in other organs. The brain plays a central role in coordinating sexual function, including the regulation of reproductive development, maturation and sexual behaviour in both sexes. In this study, we investigated sex-related differences in gene expression in the brains of breeding zebrafish (Danio rerio) to establish a greater understanding of the sex-specific physiology of the brain in lower vertebrates. The brain transcriptomic profiles of males and females were interrogated to identify the genes showing sexually dimorphic gene expression. 42 genes were differentially expressed between the sexes, from which 18 genes were over-expressed in males and 24 genes were over-expressed in females. In males, these included deiodinase, iodothyronine, type II and ribosomal protein S8, and in females, superoxide dismutase [Cu-Zn], sprouty-4, frizzled 10 and testis enhanced gene transcript. Estrogen responsive elements were found in the regulatory regions for 3 genes over-expressed in males and 7 genes over-expressed in females. We have demonstrated the existence of dimorphic patterns of gene expression in the brain of a sexually mature, non-mammalian, vertebrate model, with implications for studies into reproduction and chemical disruption of brain function.

Environmental oestrogens are widespread in the aquatic environment and cause alterations in sexual development and function in vertebrates. The molecular pathways underpinning these effects, however, remain poorly understood. In this study, we aimed at generating a mechanistic understanding of the disruptive effects of exposure to environmentally relevant concentrations of 17 alpha-ethinyloestradiol (EE(2)) on reproduction in zebrafish, by anchoring the transcriptomic alterations induced with the physiological consequences of exposure. Breeding colonies of zebrafish were exposed for a 21-day period to three concentrations of EE(2) (0.05, 0.5 and 5 ng/L) and the gonadal transcriptomic alterations induced (determined using a 17,000 oligonucleotide microarray) were analysed together with physiological effects seen on reproductive output of both males and females. Exposure to 5 ng EE(2)/L resulted in reproductive impairment characterised by a decrease in egg production, alterations in sperm quality and reduced fertilisation success. The effects seen were associated with altered expression of 114 and 131 genes in the gonads of males and females, respectively. The biological processes most affected by the exposure were protein metabolism in males and mitochondria organisation and biogenesis in females. Genes involved in the regulation of cell cycle progression, the ubiquitin system and glutathione peroxidase were affected by the EE(2) exposure and associated with the changes observed in gamete quality in both genders. In summary, we demonstrated that EE(2) exposure compromised the reproductive health of breeding zebrafish at environmentally relevant concentrations. The molecular mechanisms mediating some of these effects were identified and included those impacting processes central to gametogenesis in both males and females.

Thyroid hormones have been implicated as important regulators of teleost development. To gain a better understanding of the potential roles of the thyroid system in salmonids a genomic clone which encoded rainbow trout TR-alpha was isolated. This clone exhibited highest amino acid identity to Japanese flounder TR-alphaB (94%) and zebrafish TR-alpha1 (94%). Oligonucleotides were designed against the rainbow trout sequence and the complete coding region of Atlantic salmon TR-alpha was isolated by RACE-PCR. The Atlantic salmon sequence exhibited highest amino acid identity to rainbow trout TR-alpha (98%), Japanese flounder TR-alphaB (93%), and zebrafish TR-alpha1 (90%). Atlantic salmon TR-alpha exhibited the classic modular structure associated with members of the nuclear receptor superfamily and consisted of a divergent A/B domain while the DNA and ligand-binding domains were highly conserved to other teleost TR proteins. Temporal expression from the rainbow trout TR-alpha gene was monitored by semiquantitative RT-PCR at selected stages during rainbow trout embryonic and larval development. High levels of maternal transcripts were present at cleavage (Stage 6) which were rapidly degraded by gastrulation (Stage 13). Low levels of TR-alpha expression were then detected during organogenesis (Stages 20, 24, 26, 29, and 31). A peak in mRNA levels was observed at hatch (Stage 32) after which levels rose in a gradual manner during larval development (Stages 33, 34, 35, and 36) to reach maximal values at first feeding (Stage 37). These results suggest that the thyroid axis is functional and that embryonic and larval rainbow trout are at least capable of responding to thyroid hormones. These observations implicate the thyroid system as being an important regulator of salmonid development.

Abstract Manufactured nanoparticles (MNPs) undergo transformation immediately after they enter wastewater treatment streams and during their partitioning to sewage sludge, which is applied to agricultural soils in form of biosolids. We examined toxicogenomic responses of the model nematode Caenorhabditis elegans to pristine and transformed ZnO-MNPs (phosphatized pZnO- and sulfidized sZnO-MNPs). To account for the toxicity due to dissolved Zn, a ZnSO 4 treatment was included. Transformation of ZnO-MNPs reduced their toxicity by nearly ten-fold, while there was almost no difference in the toxicity of pristine ZnO-MNPs and ZnSO 4. This combined with the fact that far more dissolved Zn was released from ZnO- compared to pZnO- or sZnO-MNPs, suggests that dissolution of pristine ZnO-MNPs is one of the main drivers of their toxicity. Transcriptomic responses at the EC 30 for reproduction resulted in a total of 1161 differentially expressed genes. Fifty percent of the genes differentially expressed in the ZnSO 4 treatment, including the three metal responsive genes (mtl-1 , mtl-2 and numr-1), were shared among all treatments, suggesting that responses to all forms of Zn could be partially attributed to dissolved Zn. However, the toxicity and transcriptomic responses in all MNP treatments cannot be fully explained by dissolved Zn. Two of the biological pathways identified, one essential for protein biosynthesis (Aminoacyl-tRNA biosynthesis) and another associated with detoxification (ABC transporters), were shared among pristine and one or both transformed ZnO-MNPs, but not ZnSO 4. When comparing pristine and transformed ZnO-MNPs, 66% and 40% of genes were shared between ZnO-MNPs and sZnO-MNPs or pZnO-MNPs, respectively. This suggests greater similarity in transcriptomic responses between ZnO-MNPs and sZnO-MNPs, while toxicity mechanisms are more distinct for pZnO-MNPs, where 13 unique biological pathways were identified. Based on these pathways, the toxicity of pZnO-MNPs is likely to be associated with their adverse effect on digestion and metabolism. Graphical abstract Image 1 Highlights • Pristine ZnO-MNPs and ZnSO 4 show similar toxicity to Caenorhabditis elegans. • Toxicogenomic responses in all treatments are partially explained by dissolved Zn. • Transformed ZnO-MNPs show similar in toxicity but different transcriptomic responses. • Distinct toxicity pathways are indicated for the phosphatized ZnO nanoparticles. The toxicity and transcriptomics responses of transformed ZnO nanoparticles are distinct from pristine ZnO nanoparticles and only partially due to release of ions. [ABSTRACT FROM AUTHOR]

Polychaetes are frequented in toxicological studies, one reason being that some members occupy shallow burrows in sediments and are maximally exposed to the contaminants that accumulate within them. We have been studying one population of the polychaete Nereis (Hediste) diversicolor exhibiting inheritable tolerance to extreme copper contamination in estuarine sediment. Using transcriptome sequencing data we have identified a suite of genes with putative roles in metal detoxification and tolerance, and measured their regulation. Copper tolerant individuals display significantly different gene expression profiles compared to animals from a nearby population living without remarkable copper levels. Gene transcripts encoding principle copper homeostasis proteins including membrane copper ion transporters, copper ion chaperones and putative metallothionein-like proteins were significantly more abundant in tolerant animals occupying contaminated sediment. In contrast, those encoding antioxidants and cellular repair pathways were unchanged. Nontolerant animals living in contaminated sediment showed no difference in copper homeostasis-related gene expression but did have significantly elevated levels of mRNAs encoding Glutathione Peroxidase enzymes. This study represents the first use of functional genomics to investigate the copper tolerance trait in this species and provides insight into the mechanism used by these individuals to survive and flourish in conditions which are lethal to their con-specifics. [ABSTRACT FROM AUTHOR]

Background: Dietary zinc supplementation may help to promote growth, boost the immune system, protect against diabetes, and aid recovery from diarrhoea. We exploited the zebrafish (Danio rerio) gill as a unique vertebrate ion transporting epithelium model to study the time-dependent regulatory networks of gene-expression leading to homeostatic control during zinc supplementation. This organ forms a conduit for zinc uptake whilst exhibiting conservation of zinc trafficking components. Results: Fish were maintained with either zinc supplemented water (4.0 μM) and diet (2023 mg zinc kg-1) or water and diet containing Zn2+ at 0.25 μM and 233 mg zinc kg-1, respectively. Gill tissues were harvested at five time points (8 hours to 14 days) and transcriptome changes analysed in quintuplicate using a 16 K microarray with results anchored to gill Zn2+ influx and whole body nutrient composition (protein, carbohydrate, lipid, elements). The number of regulated genes increased up to day 7 but declined as the fish acclimated. In total 525 genes were regulated (having a fold-change more than 1.8 fold change and an adjusted P-value less than 0.1 which is controlling a 10% False discovery rate, FDR) by zinc supplementation, but little overlap was observed between genes regulated at successive time-points. Many genes displayed cyclic expression, typical for homeostatic control mechanisms. Annotation enrichment analysis revealed strong overrepresentation of "transcription factors", with specific association evident with "steroid hormone receptors". A suite of genes linked to "development" were also statistically overrepresented. More specifically, early regulation of genes was linked to a few key transcription factors (e.g. Mtf1, Jun, Stat1, Ppara, Gata3) and was followed by hedgehog and bone morphogenic protein signalling. Conclusions: The results suggest that zinc supplementation reactivated developmental pathways in the gill and stimulated stem cell differentiation, a response likely reflecting gill remodelling in response to its altered environment. This provides insight to the role of zinc during cell differentiation and illustrates the critical nature of maintaining zinc status. The study also highlights the importance of temporal transcriptomics analysis in order resolve the discrete elements of biological processes, such as zinc acclimation. [ABSTRACT FROM AUTHOR]

Background: Physiologically based modelling using DEBtox (dynamic energy budget in toxicology) and transcriptional profiling were used in Caenorhabditis elegans to identify how physiological modes of action, as indicated by effects on system level resource allocation were associated with changes in gene expression following exposure to three toxic chemicals: cadmium, fluoranthene (FA) and atrazine (AZ). Results: For Cd, the physiological mode of action as indicated by DEBtox model fitting was an effect on energy assimilation from food, suggesting that the transcriptional response to exposure should be dominated by changes in the expression of transcripts associated with energy metabolism and the mitochondria. While evidence for effect on genes associated with energy production were seen, an ontological analysis also indicated an effect of Cd exposure on DNA integrity and transcriptional activity. DEBtox modelling showed an effect of FA on costs for growth and reproduction (i.e. for production of new and differentiated biomass). The microarray analysis supported this effect, showing an effect of FA on protein integrity and turnover that would be expected to have consequences for rates of somatic growth. For AZ, the physiological mode of action predicted by DEBtox was increased cost for maintenance. The transcriptional analysis demonstrated that this increase resulted from effects on DNA integrity as indicated by changes in the expression of genes chromosomal repair. Conclusions: Our results have established that outputs from process based models and transcriptomics analyses can help to link mechanisms of action of toxic chemicals with resulting demographic effects. Such complimentary analyses can assist in the categorisation of chemicals for risk assessment purposes. [ABSTRACT FROM AUTHOR]

Abstract: The transcription of metal-responsive genes in the rainbow trout (Oncorhynchus mykiss) gill tissue can be used to detect effects of bioreactive metals in natural waters. Here we take advantage of an in vitro gill epithelium, which can be directly exposed to test water samples. The in vitro gill epithelial model mimics the molecular response of in vivo gill epithelial cells to waterborne contaminants. The same culture system can detect trace metals and organic waterborne contaminants. Furthermore, combining this epithelial model with transcriptomic profiling yields an extremely discriminatory biomonitoring tool able to detect and differentiate waterborne metal contaminants. The bioreactive fraction of metal in the water sample is detected using the cells naturally occurring metal sensor, metal-responsive transcription factor 1 (MTF1), which acts upon Metal Response Elements (MRE''s) in the enhancer region of metal regulated genes. Induction of the MTF1 responsive genes, metallothionein-A (MTA), metallothionein-B (MTB), and zinc transporter 1 (ZnT-1) in the cell culture was strongly dependent of the concentrations of bioreactive zinc and silver in the test water. Importantly, gene expression in cell culture reflected animal toxicity, measured as inhibition of Ca2+ and Na+ influx, in live rainbow trout exposed to the same waters. A cDNA microarray was deployed to determine the differential profiles of transcripts characteristic of exposure to silver, copper or cadmium within this in vitro system. These experiments illustrated the potential power of combining the in vitro gill model epithelium with genetic profiling for accurate characterisation and identification of bioreactive toxicants in waterborne samples. [Copyright &y& Elsevier]

We have developed a novel test system for the quantitative assessment of gene transcription. The procedure involves the use of chemiluminescent-labeled oligonucleotide probes in a hybridization protection assay (HPA) format. We have used this technology to measure changes in vitellogenin mRNA to demonstrate the impact of estrogen exposure in the juvenile fathead minnow (Pimephales promelas). Marked changes in mRNA expression were observed in response to intraperitoneal injection of 17β-estradiol demonstrating the utility of this technique for the identification and monitoring of toxic responses to xenobiotics. [ABSTRACT FROM AUTHOR]

Abstract: Trametes versicolor is an important white rot fungus of both industrial and ecological interest. Saprotrophic basidiomycetes are the major decomposition agents in woodland ecosystems, and rarely form monospecific populations, therefore interspecific mycelial interactions continually occur. Interactions have different outcomes including replacement of one species by the other or deadlock. We have made subtractive cDNA libraries to enrich for genes that are expressed when T. versicolor interacts with another saprotrophic basidiomycete, Stereum gausapatum, an interaction that results in the replacement of the latter. Expressed sequence tags (ESTs) (1920) were used for microarray analysis, and their expression compared during interaction with three different fungi: S. gausapatum (replaced by T. versicolor), Bjerkandera adusta (deadlock) and Hypholoma fasciculare (replaced T. versicolor). Expression of significantly more probes changed in the interaction between T. versicolor and S. gausapatum or B. adusta compared to H. fasciculare, suggesting a relationship between interaction outcome and changes in gene expression. [ABSTRACT FROM AUTHOR]

This study correlates sub-organismal changes with toxicological effects in earthworms (Lumbricus rubellus) exposed to copper and cadmium. Both metals reduced survival and reproduction at the highest concentration (LC50 5.11 μM Cu g-1 and 4.04 μM Cd g-1; cocoon production EC50s 5.17 μM Cu g-1 and 1.86 μM Cd g-1, all values as dry mass soil). Cadmium significantly reduced lysosomal membrane stability (at 1.86 μM Cd g-1 and higher), upregulated metallothionein gene expression (at least sevenfold in all treatments) and reduced lysosome-associated-glycoprotein gene expression. Copper did not lower lysosomal membrane stability, but did upregulate metallothionein gene expression (at 2.5 μM Cu g-1), reduce lysosome-associated-glycoprotein gene expression and gave a nonlinear pattern for mitochondrial ribosomal subunit transcript expression (reduced at 0.35 and 0.811 μM Cu g-1; higher at 2.5 μM Cu g-1). Correlation of metal body residue concentrations and cellular and molecular genetic responses with juvenile production rate confirmed a relationship for metallothionein expression, lysosomal membrane stability and cadmium tissue concentration in cadmium-exposed worms. Relationships between responses were also found for both metals. These suggested mechanisms for the interaction of cadmium and copper with specific gene products and with organelle (mitochondrial, lysosomal) functioning. [Copyright &y& Elsevier]

Polychaetes are vital for evaluating the effects of toxic metals in marine systems, and sensitive molecular biomarkers should be integral to monitoring efforts. However, the few polychaete markers that exist are inconsistent, even within the same species, failing to identify gene expression changes in metal-exposed animals incurring clear metabolic costs. Comparing previously characterised polychaete metal-responsive genes with those of another carefully selected species could identify biomarkers applicable across polychaetes. The ragworm Alitta virens (Sars, 1835) is particularly suited for such comparisons due to its dominance of fully saline coastal areas, widespread distribution, large biomass, and its phylogenetic position relative to other polychaete 'omic' resources. A transcriptome atlas for A. virens was generated and an RNASeq-qPCR screening approach was used to characterise the response to chronic exposures of environmentally relevant concentrations of copper and zinc in controlled mesocosms. Genes presenting dramatic expression changes in A. virens were compared with known metal-responsive genes in other polychaetes to identify new possible biomarkers and assess those currently used. This revealed some current markers should probably be abandoned (e.g. Atox1), while others, such as GST-Omega , should be used with caution, as different polychaete species appear to upregulate distinct GST-Omega orthologues. In addition, the comparisons give some indication of genes that are induced by metal exposure across phylogenetically divergent polychaetes, including a suite of haemoglobin subunits and linker chains that could play conserved roles in metal-stress response. Although such newly identified markers need further characterisation, they offer alternatives to current markers that are plainly insufficient. Unlabelled Image • We provide a comprehensive transcriptome for the polychaete Alitta virens. • Metal-induced genes across divergent polychaetes are identified. • Current markers should be abandoned (Atox1) or used conservatively (GST-Omega). • Haemoglobins induced in divergent metal-exposed polychaetes may be better markers. [ABSTRACT FROM AUTHOR]

During the last few years, there has been an alarming increase in the amount of nickel (Ni) being released into the environment, primarily due to its use in the production of stainless steel but also from other sources such as batteries manufacturing and consequent disposal. The established biotic ligand models provide precise estimates for Ni bioavailability, in contrast, studies describing the mechanisms underpinning toxicological effect of Ni are scarce. This study exploits RNA-seq to determine the transcriptomic responses of isopods using Porcellionides pruinosus as an example of a terrestrial metal-resistant woodlouse. Furthermore, the recently proposed model for Ni adverse outcome pathways (Ni-AOP) presents an unprecedented opportunity to fit isopod responses to Ni toxicity and define Porcellionides pruinosus as a metalomic model. Prior to this study, P. pruinosus represented an important environmental sentinel, though lacking genetic/omic data. The reference transcriptome generated here thus represents a major advance and a novel resource. A detailed annotation of the transcripts obtained is presented together with the homology to genes/gene products from Metazoan and Arthropoda phylum, Gene Ontology (GO) classification, clusters of orthologous groups (COG) and assignment to KEGG metabolic pathways. The differential gene expression comparison was determined in response to nickel (Ni) exposure and used to derive the enriched pathways and processes. It revealed a significant impact on ion trafficking and storage, oxidative stress, neurotoxicity, reproduction impairment, genetics and epigenetics. Many of the processes observed support the current Ni-AOP although the data highlights that the current model can be improved by including epigenetic endpoints, which represents key chronic risks under a scenario of Ni toxicity. • Novel transcriptome for the cosmopolitan superspecies Porcellionides pruinosus. • Ni impacts methylation genes in P. pruinosus. • Phylogenetic homology of this terrestrial isopod transcriptome to other related groups. [ABSTRACT FROM AUTHOR]